The active site gorge of acetyicholinesterase (AChE) is composed of two separate ligand binding sites, an acylation site at the base of the gorge and a peripheral site at the opening of the gorge some 15 Angstrom from the acylation site. Inhibitors can bind to either site exclusively or to both sites simultaneously. Fasciculins (snake venom neurotoxins) are unique peripheral site inhibitors in that they produce an allosteric effect at the acylation site in addition to steric blockade at the mouth of the gorge. Crystal structures of both mouse and Torpedo AChE complexed with fasciculin II show that loop 2 is responsible for blocking the opening of the gorge while loop 1 makes contacts along the AChE omega loop (C67-C94, Torpedo numbering). Organophosphates are potent irreversible inhibitors of AChE and are commonly encountered in the form of pesticides. We hypothesize that Fas inhibits AChE through an allosteric mechanism mediated by the extensive interactions between loop 1 of fasciculin and the omega-loop of AChE. We propose that these interactions differentiate Fas from other peripheral site inhibitors. We will test this hypothesis by pursuing the following three specific aims: 1) Express HuAChE omega loop site-directed mutants and measure the allosteric effect upon wtFas binding. 2) Construct fasciculin site-directed mutants of residues in loop 1 and those in loop 2 that make contact with the omega loop. 3) Reanalyze previous Fas inhibition studies to clarify the extent to which Fas inhibits substrate hydrolysis. Once the allosteric mechanism is characterized, we will be prepared to design small molecule inhibitors that allosterically inhibit organophosphorylation.